Regulatory proteins have a central role in health and disease: DNA- binding proteins control development, differentiation and cell growth. Many DNA-binding proteins can be grouped into families that use related structural motifs for recognition, and focusing on these key motifs provides a useful strategy for studying protein-DNA recognition and gene regulation. The homeodomain - which uses a helix-turn-helix unit and an N-terminal arm for recognition - is one of the largest and most important families of eukaryotic regulatory proteins. Hundreds of homedomain proteins have been characterized, and these proteins play critical roles in development, specification of cell type, and tissue-specific gene expression. Although there has been tremendous progress in the structural analysis of protein-DNA complexes, we still do not understand the fundamental principles of recognition. Our goal is to use the homeodomain as a model structural system for the detailed physical/chemical analysis of protein- DNA interactions. We will continue our structural studies of homeodomain- DNA interactions, analyzing these complexes at higher resolution and determining the effects of mutating key residues. Related crystallographic studies will focus on: 1) the POU domain, which contains a homeodomain, a linker region, and a POU-specific domain that has structural similarities with the lambda repressor and on 2) the paired domain, which plays important roles in human development and frequently occurs in conjunction with homeodomains. By focusing our efforts on a set of carefully chosen model systems we hope that we can develop a coherent body of biochemical and structural data that may lead to a fundamental advance in our understanding of protein-DNA recognition and its role in gene regulation.